Banking of Cells, Tissues, and Organs at Low Temperatures

Pegg, D.E.

doi:10.1007/978-1-4757-9933-0_6

Cited by 8 publications

(6 citation statements)

References 47 publications

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“…This assumption is of central importance to the field of cryobiology, for it has led to certain hypotheses about the modes of protection and injury during freezing (Lovelock, 1953;Meryman, 1971 b) and to certain rationales in attempts to preserve the complex cellular systems that comprise tissues and organs (Pegg, 1970). If the assumption is wrong, then so too must be portions of the hypotheses.…”

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confidence: 97%

Permeability of the bovine red cell to glycerol in hyperosmotic solutions at various temperatures

1974

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Summary.A central tenet in cryobiology is that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to estimate the amount of solute that has permeated a cell prior to freezing. The amount in bovine red cells was estimated from the flux equationSolving the equation required estimates of Pr, the permeability constant for the solute. Estimates for glycerol in bovine red cells were made in two ways: (1) by measuring the time to 50% hemolysis of red cells suspended in isosmotic or hyperosmotic (1 to 3 M) solutions of glycerol that were hypotonic with respect to NaC1, and (2) by measuring the time required for red cells in hyperosmotic solutions of glycerol in isotonic salinebuffer to become susceptible to osmotic shock upon 10-fold dilution with isotonic saline-buffer. The measurements were made at 0, 10, 15 and 20 ~ The values by the second technique ranged from 2.3 • 10 -6 cm/min to 2.7 x 10 -6 cm/min at 20 ~ depending on the concentration of glycerol. The values by the first technique were 0 to 30 % lower. Both techniques yielded about the same activation energy for permeation between 0 and 20 ~ 21 kcal/mole. This is equivalent to a halving of the permeation rate for every 5 ~ drop in temperature.Expressing the flux equation in the formulation of irreversible thermodynamics changed the value of P by less than 10 %, probably because a, the reflection coefficient, is 0.95 at 25 ~ Expressing the driving force as the difference in molality or osmolality of glycerol, rather than as the difference in activity, however, had somewhat greater effects on the numerical values of P, but had no effect on the activation energy.It is concluded that estimates of P based on differences in activities and on the osmotic shock technique are the least subject to error.

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confidence: 97%

Permeability of the bovine red cell to glycerol in hyperosmotic solutions at various temperatures

1974

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“…1-4 must, before reaching the intracellular nucleation temperature, be reduced to the equilibrium water content given by ln[V/(V + n2v,0)] = (L,/RX1/273 -1/T). [5] …”

Section: Introductionmentioning

confidence: 99%

“…1-4 must, before reaching the intracellular nucleation temperature, be reduced to the equilibrium water content given by ln[V/(V + n2v,0)] = (L,/RX1/273 -1/T). [5] Details of the derivation of these equations and of the underlying assumptions have been published (13 (20,21) and assume a nucleation temperature of -40°, calculations from Eqs. 1-4 indicate that avoidance of intracellular freezing would require cooling rates of -1°/min or less (22).…”

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confidence: 99%

“…However, long-term low-temperature storage requires temperatures below -700; and confirmed instances of organs surviving cooling to such temperatures are rare-most are irreversibly damaged below -200 (5,6). Nevertheless, recent reports of the successful freezing of isolated islets of Langerhans (7,8) and recent achievements in the freezing of early mammalian embryos (9)(10)(11)(12) encouraged us to attempt to freeze the intact fetal pancreas.…”

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confidence: 99%

“…Pancreases were removed from 161'-to 17w-day fetuses of Sprague-Dawley rats and placed in solutions of saline (8. (5,6,29,30). However, these initial experiments yielded poor survival, and so we adopted the following procedure which will be referred to as standard.…”

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confidence: 99%

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Survival of fetal rat pancreases frozen to -78 and -196 degrees.

Mazur¹,

Kemp²,

Miller³

1976

Proc. Natl. Acad. Sci. U.S.A.

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Transplantation of pancreases may have clinical utility in the treatment of diabetes, for it has been shown that chemically induced diabetes in rats can be reversed by the transplantation of 2 four syngeneic fetal pancreases. Allogeneic transplants produce serious It has recently been shown that diabetes mellitus induced in rats with streptozotocin can be reversed by implantation of four or more pancreases from syngeneic 16-to 18-day fetal rats (1-4). The ability to establish banks of frozen, tissue-typed, fetal pancreases might facilitate similar procedures in allogeneic animals, including man.However, long-term low-temperature storage requires temperatures below -700; and confirmed instances of organs surviving cooling to such temperatures are rare-most are irreversibly damaged below -200 (5, 6). Nevertheless, recent reports of the successful freezing of isolated islets of Langerhans (7,8) and recent achievements in the freezing of early mammalian embryos (9-12) encouraged us to attempt to freeze the intact fetal pancreas.The critical cryobiological factors affecting cell survival are: (i) cooling rate, (Hi) warming rate, (iii) concentration of protective additive, (iv) extent to which additives have permeated the cell prior to freezing, and (v) osmotic problems engendered by attempts to remove the additive after thawing. Cells cooled too rapidly are almost always killed, with death being a result of intracellular freezing. Theoretical considerations allow one to define this critical cooling rate (13-15), and the theory has been confirmed by experiments in red blood cells, yeast, tissue cultured cells, and embryos (16)(17)(18)(19).Even in the presence of extracellular ice, cells initially supercool, and the resulting difference in the chemical potentials of intra-and extracellular water causes water to flow out of them (13). To avoid lethal intracellular freezing, the cells must be cooled slowly enough to permit them to come into equilibrium with external ice before reaching their nucleation temperature. The rate of exosmosis during freezing is described by four simultaneous equations, namely dV/dt = (LPART lnPe/lpi)lvi0 [1] where V is the volume of cell water, t is time, Lp the hydraulic conductivity, A the cell surface area, and v10 the molar volume of water. The symbols Pe and pi refer to the vapor pressure of extracellular and intracellular water, respectively, whereT is temperature, n2 is osmoles of solute, and Lf is the latent heat of fusion. R is the gas constant.Time and temperature are related by the cooling rate, which, if linear, is given by dT/dt = B [3] and finally, Lp is related to temperature by the expression[4] where (Lp)g is the permeability coefficient to water at a known temperature, Tg, and b is its temperature coefficient.To avoid intracellular freezing, the water content of the cell given by Eqs. 1-4 must, before reaching the intracellular nucleation temperature, be reduced to the equilibrium water content given by ln[V/(V + n2v,0)] = (L,/RX1/273 -1/T). [5]

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